Expression library immunization

ABSTRACT

A general method for vaccinating against any pathogen is presented. The method utilizes expression library immunization, where an animal is inoculated with an expression library constructed from fragmented genomic DNA of the pathogen. All potential epitopes of the pathogen&#39;s proteins are encoded in its DNA, and genetic immunization is used to directly introduce one or more expression library clones to the immune system, producing an immune response to the encoded protein. Inoculation of expression libraries representing portions of the Mycoplasma pulmonis genome was shown to protect mice from subsequent challenge by this natural pathogen. Protection against Listeria.

The application is a divisional of patent application Ser. No.08/421,155 filed Apr. 7, 1995 now issued as U.S. Pat. No. 5,703,057.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to methods for screening and obtainingvaccines generated from administration of expression librariesconstructed from a pathogen genome. The method further includesidentification of one or more antigenic plasmids that will elicit animmune response that is protective against pathogen challenge subsequentto inducing an in vivo immunogenic response. Also included in theinvention are particular vaccine compositions protective againstListeria and Mycoplasma.

2. Description of Related Art

While vaccination is one of the most cost-effective medical methods forsaving lives, vaccines have not been developed for many of the mostserious human diseases, including respiratory syncytial virus (RSV),pneumonia caused by Streptococcus pneumoniae, and diarrhea caused byrotavirus and Shigella. As is evident with the HIV epidemic, theincrease in tuberculosis and the endemic spread of malaria and otherparasitic diseases, there is an increasing need to develop effectivevaccines, yet for many of these pathogens daunting scientific problemshave arisen. For example, the influenza virus is notorious for antigenicdrift so that new vaccines are constantly being developed; researchefforts continue in attempts to identify effective vaccines for rabies(Xiang, et al, 1994), herpes (Rouse, 1995); tuberculosis (Lowrie, et al,1994); HIV (Coney, et al, 1994) as well as many other diseases ofimportance in developed and undeveloped countries. Yet there exists norelatively rapid, yet alone systematic, means of identifying aneffective vaccine, much less a reasonable assurance that, onceidentified, the vaccine will be broadly responsive to pathogenchallenge.

Many currently used vaccines are composed of live/attenuated pathogens(Ada, 1991) which when inoculated infect cells and elicit a broad immuneresponse in the host. While highly detailed knowledge of thepathobiology is not necessary, at the very least isolation andidentification of the pathogen is required. Live vaccines are oftensuperior to antigen or subunit vaccines because of their tendency toelicit a broad level protective response; however, serious disadvantagesin using such vaccines include the risk of a vaccine-induced infection,problems with producing and storing the vaccine, and failure to engenderany immune response; for example, where antigen presentation is limited.Perhaps the most troubling aspect of using live vaccines is thepropensity for actually causing the disease against which protection isintended. Past experience with some of the polio and measles vaccineshas demonstrated that this may be a serious risk.

An alternative to the use of live/attenuated pathogen vaccines is to useantibodies to single proteins or to a limited number of proteinsassociated only with the pathogen. Polyclonal or monoclonal antibodiesare readily produced with the aid of modern hybridoma technology,although these techniques are relatively expensive and time consuming.There is also no assurance that antibodies produced in response to anantigen will provide protection against the pathogen providing theantigen; consequently, it may be necessary to test a large number ofantigens isolated from a pathogen. Ultimately, no single antigen mayprove effective as a vaccine because the ability of subunit or killedvaccine preparations to elicit a broad immune response is generallyquite limited.

Certain disadvantages of conventional vaccines are overcome by usingwhat is called "genetic immunization" (Tang, 1992). This technologyinvolves inoculating simple, naked plasmid DNA encoding a pathogenprotein into the cells of the host. The pathogen's antigens are producedintracellularly and, depending on the attached targeting signals, can bedirected toward major histocompatibility complex (MHC) class I or IIpresentation (Ulmer, et al, 1993; Wang, et al, 1993). Risk of infectionis essentially eliminated and the DNA can be delivered to cells notnormally infected by the pathogen. Compared to conventional vaccines,the production of genetic vaccines is straightforward and DNA isconsiderably more stable than proteinaceous or live/attenuated vaccines.Genetic immunization (a.k.a. DNA, polynucleotide etc. immunization) withspecific genes has shown promise in several model systems of pathogenicdisease (Davis, et al, 1993; Conry, et al, 1994; Xiang, et al, 1994),and a few natural systems (Cox, et al, 1993; Fynan, et al, 1993). Use ofDNA (or RNA) thus overcomes some of the problems encountered when ananimal is presented directly with an antigen.

However, despite promising initial results with genetic vaccination,there remains the more basic and unsolved problem of identifying theparticular gene or genes of the pathogen that will express an immunogencapable of priming the immune system for rapid and protective responseto pathogen challenge. Certain non-viral pathogens and some viruses havevery large genomes; for example, protozoa genomes contain up to about10⁸ nucleotides, thus posing an expensive and time-consuming analyticalchallenge to identify or isolate effective immunogenic antigens. Thesolution to this problem to date has been to extensively study thepathobiology of the host-pathogen interaction to isolate the proteinreacted to by the host during infection. And even with identification ofa gene or subunit that elicits a protective immune response, there maystill be lacking strong protection because of lack of broad response tothe encoded polypeptide.

Significantly, the time and money to identify and develop a vaccinewould be greatly reduced if there were available simple, systematic andrapid ways to identify vaccines for specific pathogens without havingfirst to determine at least the fundamental biological properties of thepathogen. Even more important would be vaccines that are broadlyeffective without any danger of causing the disease against which it isintended to protect.

SUMMARY OF THE INVENTION

The present invention addresses one or more of these and other drawbacksinherent in the prior art by providing novel methods of generating andidentifying effective vaccines. The vaccines stimulate a broadprotective response in a manner similar to that generated bylive/attenuated vaccines without the inherent disadvantage ofpotentially causing the disease associated with the pathogen. Theinvention also includes novel antigenic polypeptides encoded by DNAplasmid vaccines, kits that include antigenic pathogen polypeptides,plasmid vaccines or antibodies generated from the pathogen polypeptides.

The invention, in general terms, arises from the inventors' success indeveloping a way to present to an animal a major number of antigenicdeterminants encoded by the genome of any pathogen. The DNA of thepathogen is fragmented, ligated into expression vectors and clonedsub-libraries (sibs) of the expression library are inoculated into ananimal. Sib library is understood to be a portion of a parental librarythat may or may not have overlapping members with other sibs of the samelibrary. "Sibbing" as used herein is understood to mean the partitioningof a parental library into sequential subsets. Challenge by the pathogenreveals which animals are protected and consequently which portions ofthe sib expression library have protective effect. Sibbing methods maythen be used to identify the individual or combinations of plasmids thatare responsible for the protection. It is then possible to preparevaccines from the plasmids, and to identify the polypeptides encoded bythe protective plasmid vectors. One may also use well known methods togenerate polyclonal or monoclonal antibodies to identified immunogensand use these as vaccines.

The invention is particularly directed to methods of vaccination. Acloned expression library is prepared from fragmented genomic DNA of apathogen, or may be prepared from cDNA of pathogen RNA. One thenintroduces one or more of the library clones into an animal so as toinduce an immune response against one or more of the antigens that areencoded by one or more of the clones. Subsequently, one may isolate theparticular clone or clones responsible for producing an antigen thatinduces an immune response, obtain the antibodies, if any, generated inresponse to one or more of the clones, or formulate vaccine compositionsfrom the various libraries or sib libraries that induce an immuneresponse.

The inventors' method allows rapid screening for potential vaccines. Italso allows discovery of new vaccines that would not be detected byconventional approaches. For such screening, one first selects oridentifies a particular pathogen to which a protective vaccine isdesired. For example, the method has been applied to a mycoplasm,Mycoplasma pulmonis, and to a eubacterium, Listeria monocytogenes. Thesepathogens are but two examples of the wide range of pathogens that mightbe screened; the method is equally adaptable to screen for vaccines forHIV, malaria, mycoplasma, tuberculosis, respiratory syncytial virus, andconjugated pneumoccus; all pathogens for which there are no effectivevaccines. One may also screen for alternative and improved vaccines fordiseases such as smallpox and polio. Nor is the method limitednecessarily to viruses and bacteria. Use of any pathogen iscontemplated, including protozoa, yeast, fungi, worms or prions. It isonly necessary to obtain genomic material e.g., DNA or RNA or, in thecase of prions, because they are proteins, to isolate or synthesize aDNA that encodes the prion.

After selecting the pathogen(s) to which a vaccine is desired, one thenobtains a genomic or cDNA (or RNA) sample which is subsequentlyfragmented, for example by physical fragmentation or, preferably, byenzymatic cleavage, i.e. use of restriction endonucleases. Fragmentationmethods are well known to those skilled in the art and may be varied toobtain segments (by use of different restriction endonucleases orcombinations and digestion times) differing in size and composition.

After fragmentation of the DNA, an expression library is prepared.Preparation of such libraries is relatively straightforward and can beperformed by well known methods. Standard cloning vectors such as Puc118may be employed which have an ampicillin selectable marker and,preferably ori and a CMV promoter. Bacteria are then transformed withthe vectors, for example, E. coli or Salmonella or other suitablebacterial host. Identified transformants are cultured by standardprocedures and the plasmid DNA isolated by such methods aschromatographic or organic separation. A series of plasmids have beenconstructed which allow cloning each library into a site which candirect the foreign protein to MHCI or II. These plasmids are shown inFIG. 1.

For prions, a relatively rare class of pathogens, a preferred method isto determine the amino acid sequence of the protein and synthesize theencoding DNA, or, alternatively, isolate encoding DNA from infectedcells. The prion-encoding DNA is then fragmented and used to prepare anexpression library in a manner analogous to that of RNA, cDNA or genomicDNA.

An important aspect of the invention is the preparation of arepresentative expression library from pathogen DNA. Mycoplasma pulmonisis one example of a pathogen. MP has a genome size of ˜1×10⁶ bp. Two ofnine sib libraries provided protective and were thus identified ascandidates for effective vaccine protection. Each sib library had ˜3×10³members, of which only ˜500 should be expressing natural open-readingframes. That two libraries protected indicated that several plasmids orcombinations of plasmids were vaccine candidates. It appears likely thata minimal number of plasmids will provide useful protection. Tests oftwo smaller MP libraries of ˜70 members derived from MP2.3 showed littleor no protection, from which it was concluded that the protectiveplasmid(s) are located in other sub-libraries of MP2.3. The inventorshave shown that the disclosed expression libraries can be used directlyas vaccines or partitioned in various ways to isolate individual orcombinations of plasmids that are protective. In this way the ELItechnology is a practical vaccine discovery approach, even for proteinsubunit vaccines.

As mentioned, DNA is fragmented either physically or, for example, byrestriction enzymes, to produce relatively small pieces, preferably onthe order of 100 to 1,000 base pairs. For smaller genomes, severalhundred base pairs are preferable, for example, 400 hundred base pairs;however, larger genomes, such as found in Pseudomonas or E. coli mightinitially be fragmented into somewhat larger sizes, for example,3000-4000 bp. Except in the case of extremely small genomes, a preferredmethod of practice is to prepare sib libraries from the main expressionlibrary. For example, a library including DNA mean fragment sizes ofapproximately 400 bp are preferably sibbed into sublibraries containingapproximately 3000 transformants, i.e. approximately 21 sublibraries. Asmaller number of sublibraries, or sib libraries, is preferred forsmaller genomes, such as mycoplasma, where a 3000 member library isachieved with approximately 9 sib libraries.

Of course it is technically feasible to apply ELI to any pathogens withlarger genomes. Genomes smaller than Mycoplasm and Listeria, many ofwhich have genomes up to 100-fold smaller than Mycoplasm, arewell-suited for application of the disclosed methods of identifying andisolating immunogens. These pathogens include HIV, known to have anexceptional number of variants and which is therefore an excellentcandidate for screening with the ELI.

Genetic immunization with the ELI expression library reproduces the sameantigens induced by a live/attenuated pathogen, i.e., the entire genome.Additionally, and importantly, the method is such as to allowpresentation of new determinants that are normally hidden by the biologyof the pathogen. The new ELI method combines the advantages of geneticimmunization without the necessity of discovering a single protectivegene or foreknowledge of the pathogen's biology.

Once expression libraries have been prepared for a given pathogen andDNA plasmid libraries isolated, one will inoculate a mammal with a DNAplasmid library. The library may be inoculated into the animal in anyone of several different methods that have been shown effective forgenetic immunization; for example, gene gun or needle injection intomuscle or skin or by oral administration. The gene gun technique usedfor ELI is thought to be approximately 1,000- to 10,000-fold moreefficient than injection of naked DNA (Fynan, et al. (1993)).Alternatively, others e.g Ulmer et al. (1993), have indicated thegenetic immunization by direct DNA injection may be performed withsimilar efficiency as the gene gun. The inventors have found that geneadministration by either method produces qualitatively identicalimmunization.

The inventors' method has broad application as a screening method foridentifying vaccines. Briefly, this involves preparing an expressionlibrary from fragmented genomic nucleic acid, isolating plasmid DNA andimmunizing with at least a portion of the library. A protective effectis indicated when an animal is challenged with the pathogen from whichthe genomic material was obtained.

The inventors have found that mammalian genes fused to the pathogen DNAappears to facilitate expression in the mammalian cell. In preferredembodiments, a mammalian gene such as that encoding human growth hormoneis fused with the DNA; however, other genes would be suitable, includinga-trypsin, ubiquitin or signal sequences. The inventors have found thatfusion of nonmammalian pathogen sequences to mammalian genes increasesthe amount of antigen available to the immune system. This may arisebecause of increasing antigenic recognition or targeting to componentsin the cell.

The inventors have found that the maximum expression by geneticimmunization (ELI) is obtained when about 2.5 mg is introduced per siteof inoculation when gene gun inoculation is used. Different amounts maybe required if other methods of introduction are employed. The inventorshave also determined that the lowest amount of DNA that produces animmune response is between about 0.1 and 1 ng. (FIG. 2). This was shownby injecting various amounts of human growth hormone DNA into mice andmonitoring antibody production. Antibody was detectable with 1 ng. Itwas thus predicted that the maximum complexity represented byinoculating 10 mg (in four sites) of a library under these conditionssuch that an immune response would be generated comprising approximately1×10⁴ clones (or 1/6×1×10⁴ =1.5×10³ expressing clones). This indicatedthat approximately 1×10⁴ clones could be included in each sib library.An inoculation of approximately 10 mg of DNA would introduce theequivalent of approximately 10⁹ bacterial genomes into the host in ahighly immunogenic form. This exceeds by several orders of magnitude thenumber of genome equivalents generally necessary to produce infection ina host. For example, mycoplasma can produce an infection when inoculatedat 10³ to 10⁶ organisms per animal. These results were an importantaspect of the invention because there was a real question whether thesepredictions concerning the number of plasmids that could be injectedwould actually be feasible. Had large libraries not been shown to beprotective, there would have been little value to the disclosed methods.

Genetic immunization procedures are now well established and well knownin the art. One or more inocula of the library aliquots may be employedfor immunization. Likewise, vaccine compositions may vary widely, toinclude for example various adjuvants.

ELI is expected to elicit response of both arms of the immune system.Extracellular antigens are largely presented through MHCII proteins andproduce a humoral defense, i.e., circulating antibodies against proteinsof the pathogen. Intracellular pathogens present proteins through adifferent pathway that goes through the endoplasmic reticulum and ontoMHCI proteins to elicit the cellular arm of the immune system. For manypathogens, the relative importance of the two arms in protection is notknown and the two systems may crossover in macrophages and throughnatural immunity, e.g., natural killer cells. Use of appropriate vectorsmay cause one or both of the immune responses to be favored.

The inventors have shown that ELI will produce a vaccine without knowingwhat specific protein(s) is responsible for eliciting protection. Inthis sense ELI mimics live/attenuated vaccines. Another advantage overconventional vaccines is that peptides may be presented to the immunesystem with ELI which are normally hidden by the pathogen's biology orimmune-avoidance mechanisms. Unlike live/attenuated vaccines where thestoichiometry of the pathogen's antigens is fixed, the composition ofthe library can be modified at will to allow introduction of only themost effective antigens at varied levels. By using ELI, the site ofinoculation can be controlled allowing cells not normally infected topresent antigens. In addition, use of other fusion proteins than hGHshould allow antigens to be targeted to specific presentation pathways.

The effective protection against mycoplasm demonstrated by the inventorsindicated both humoral and cellular responses were elicited. Previouswork demonstrated that anti-mycoplasma antibodies can protect miceagainst infection (Tayler, et al, 1981), yet passive transfer ofantibody does not protect rats (Davidson, et al, 1982). Immune spleencells, however, can transfer protection in syngeneic rats (Cassell,1982; Lai, et al, 1991). It has also been demonstrated in mice thataugmented natural killer cells or secreted interferon gamma-activatedmacrophages can kill mycoplasma (Lai, et al, 1990). Given thisuncertainty about the relative contributions to resistance and the factthat both responses were elicited with the library inoculations, thestrong protection does not appear to arise primarily from one or theother immune arm. This aspect is very attractive, as it indicates abroad protective response from ELI immunization.

ELI is expected to elicit immunity against any organisms requiringeither arm of the immune system because it activates both humoral andcellular immune responses. The protection afforded is therefore quitebroad and is expected to provide superior protection compared withsingle antigen or antigen-encoding DNA.

The invention also contemplates that the disclosed DNA libraries or thepeptides encoded by identified protective DNA plasmids will be useful indeveloping a wide range of vaccines and will also be useful in certainmethods of cancer treatment. Cancer treatment methods, including vaccinedevelopment are another aspect of the present invention. Additionally, avariety of in vitro and in vivo assay protocols are facilitated as aresult of the novel compositions disclosed herein. In addition togenerating an immune response in an animal, and particularly in a human,the peptides may also be used as immunogens to generate anti-peptideantibodies, which themselves have many uses, not least of which is thedetection of pathogen related peptides, or peptide fragments thereof, indiagnostic tests and kits based upon immunological binding assays).

Therefore, one contemplated use for the pathogen peptides concerns theiruse in methods for detecting the presence of a pathogen within a sample.These methods include contacting a sample suspected of containing apathogen with a peptide or composition in accordance with the presentinvention under conditions effective to allow the peptide(s) to form acomplex with pathogen-related peptides contained in the sample. One thendetects the presence of the complex by detecting the presence of thepeptide(s) within the complex, e.g., by either originally usingradiolabeled peptides or by subsequently employing anti-peptideantibodies and standard secondary antibody detection techniques.

The peptides, or multimers thereof, may be dispersed in any one of themany pharmacologically-acceptable vehicles known in the art andparticularly exemplified herein. As such, the peptides may beencapsulated within liposomes or incorporated in a biocompatible coatingdesigned for slow-release. The preparation and use of appropriatetherapeutic formulations will be known to those of skill in the art inlight of the present disclosure. The peptides may also be used as partof a prophylactic regimen designed to prevent, or protect against,disease pathogens, possible cancer progression and/or metastasis and maythus be formulated as a vaccine.

The present invention also provides methods for identifying specificpathogen peptides, which methods comprise contacting the cells suspectedof containing such polypeptides with an immunologically effective amountof a composition comprising one or more specific anti-peptide antibodiesdisclosed herein.

In another aspect, the present invention contemplates a diagnostic kitfor screening samples suspected of containing pathogen polypeptides, orcells producing such polypeptides. Said kit can contain a peptide orantibody of the present invention. The kit can contain reagents fordetecting an interaction between an agent and a peptide or antibody ofthe present invention. The provided reagent can be radio-,fluorescently- or enzymatically-labeled. The kit can contain a knownradiolabeled agent capable of binding or interacting with a peptide orantibody of the present invention.

In another aspect, the present invention contemplates a diagnostic kitfor detecting mycoplasm or Listeria polypeptides. The kit comprisesreagents capable of detecting such peptides. The provided reagent mayalso be radio-, enzymatically-, or fluorescently-labeled. The kit cancontain a radiolabeled peptide capable of binding to or interacting witha mycoplasma or Listeria polypeptide, or, preferably, may contain aradiolabeled antibody capable of binding to or interacting with apeptide of the present invention. The kit can contain a polynucleotideprobe that encodes a peptide of the present invention or any of theircomplements. The kit can contain an antibody immunoreactive with apeptide of the present invention.

The reagent of the kit can be provided as a liquid solution, attached toa solid support or as a dried powder. Preferably, when the reagent isprovided in a liquid solution, the liquid solution is an aqueoussolution. When the reagent provided is attached to a solid support, thesolid support can be chromatograph media, a test plate having aplurality of wells, or a microscope slide. When the reagent provided isa dry powder, the powder can be reconstituted by the addition of asuitable solvent, optionally provided.

In still further embodiments, the present invention concernsimmunodetection methods and associated kits. It is proposed that thepeptides of the present invention may be employed to detect antibodieshaving reactivity therewith, or, alternatively, antibodies prepared inaccordance with the present invention, may be employed to detectpathogen polypeptides. In general, these methods will include firstobtaining a sample suspected of containing such a protein, peptide orantibody, contacting the sample with an antibody or peptide inaccordance with the present invention, as the case may be, underconditions effective to allow the formation of an immunocomplex, andthen detecting the presence of the immunocomplex.

In general, the detection of immunocomplex formation is quite well knownin the art and may be achieved through the application of numerousapproaches. For example, the present invention contemplates theapplication of ELISA, RIA, immunoblot (e.g., dot blot), indirectimmunofluorescence techniques and the like. Generally, immunocomplexformation will be detected through the use of a label, such as aradiolabel or an enzyme tag (such as alkaline phosphatase, horseradishperoxidase, or the like). Of course, one may find additional advantagesthrough the use of a secondary binding ligand such as a second antibodyor a biotin/avidin ligand binding arrangement, as is known in the art.

For diagnostic purposes, it is proposed that virtually any samplesuspected of comprising either the mycoplasma or Listeria or antibodysought to be detected, as the case may be, may be employed. Exemplarysamples include clinical samples obtained from a patient or animal suchas blood or serum samples, ear swabs, sputum samples, middle ear fluidor even perhaps urine samples may be employed. Furthermore, it iscontemplated that such embodiments may have application to non-clinicalsamples, such as in the titering of antigen or antibody samples, in theselection of hybridomas, and the like.

In related embodiments, the present invention contemplates thepreparation of kits that may be employed to detect the presence ofpathogen peptides and/or antibodies in a sample. Generally speaking,kits in accordance with the present invention will include a suitableantigenic peptide, e.g from mycoplasma or Listeria, or an antibodydirected against such a protein or peptide, together with animmunodetection reagent and a means for containing the antibody orantigen and reagent. The immunodetection reagent will typically comprisea label associated with the antibody or antigen, or associated with asecondary binding ligand. Exemplary ligands might include a secondaryantibody directed against the first antibody or antigen or a biotin oravidin (or streptavidin) ligand having an associated label. Of course,as noted above, a number of exemplary labels are known in the art andall such labels may be employed in connection with the presentinvention.

The container means will generally include a vial into which theantibody, antigen or detection reagent may be placed, and preferablysuitably aliquotted. The kits of the present invention will alsotypically include a means for containing the antibody, antigen, andreagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Examples of ELI vectors to direct antigens for different MHCclass presentation. Secreted antigens are expected to favor MHC classpresentation and antibody production. Cytoplasmic orproteasomal-directed antigens should favor MHC class I presentation andCD8+ cytotoxic T lymphocyte activation.

FIG. 2. hGH antibodies produced by genetic immunization with variedamounts of CMV-GH plasmid. 5-6 week old female Balb/C mice wereinoculated every two weeks in 2 shots with the gene gun using theindicated amounts of CMV-GH plasmid. Each inoculum under 1 mg wasbalanced to 1 mg with CMV-LUC, a luciferase expression plasmid.Inoculations indicates samples of sera collected 10 days after theindicated CMV-GH inoculation. The entire set of sera along withpre-immune sera was tested by b-galactosidase ELISA. Anti-hGH Antibodies(betagal lumens) represents the ELISA lumens produced by each sampleminus the pre-immune background. The black and striped bars for eachdifferent amount of CMV-GH represents an individual mouse tested.

FIG. 3. Vectors used for expression library immunization (ELI).CMV-GH-F1&3 was derived from CMV-GH in which the cytomegaloviruspromoter drives expression of the genomic human growth hormone (hGH)gene. CMV-GH-F1&3 differs from the hGH sequence by substitution of aBamHI site 3' to the BglII site such that restriction fragments havingGATC 5' overhangs (i e. MboI fragments) can be inserted into BamHI orBglII in two different coding frames. TAA stop codons were inserted inall three coding frames 3' to both insertion sites to stop translationof any inserts lacking their own stop codons. CMV-GH-F2 was constructedsimilarly by knocking out the BglII site in hGH and inserting a BamHI inframe 2.

FIG. 4A. MP titers from the lungs of ELI-immunized, MP-challenged mice.5-6 week old Balb/C female mice were immunized 4 times with 10 mg ofMP1.1 and MP2.3 on day 1 and with 5 mg on day 8, 21, and 52 andchallenged with the indicated number of MP (MP Inoculum) 12 days later(Lai et al, 1991). CMV-GH and listeria library mice were immunized on asimilar schedule and challenged at the same time. The listeria librarymice consists of a 3000 transformant library constructed into CMV-GH-F2using MboI-digested Listeria monocytogenes genomic DNA. The mice weresacrificed 14 days after challenge and lung lavage and lung sectioningwas performed. MP Titers from the lungs of ELI-immunized, MP-challengedmice. MP CFUs from Lung represents the total number of MP from eachgroup of mice as calculated from counting MP grown on plates from serialdilutions of lung lavages from the mice.

FIG. 4B. Histopathology of ELI immunized, MP-challenged mice. Lungsections from mice immunized with the indicated plasmids were stainedand the degree of histopathologic lesions induced by MP infection wasassessed and scored with a histopathology index. An index of 1.0represents the maximal number of lesions observed in infected mice. Anindex of 0 indicates normal morphology. Each bar represents the meanfrom 2 to 4 mice. Error bars represent the standard deviation for eachgroup.

FIG. 5. MP titers from the lungs of mice 30 days after the start ofimmunization. 5-6 week old male Balb/C mice were immunized with 6 mg ofCMV-GH-F2 or MP2.3 on day 1 and 3 mg on day 15, and 22. The mice werechallenged with the indicated MP inoculum on day 30 and lung lavageswere performed on day 44. MP CFUs from Lung represents the total numberof MP from each group of mice as calculated from counting MP grown onplates from serial dilutions of lung lavages from the mice. Each barrepresents the mean for 3 mice. Error bars represent the standarddeviation for each group.

FIG. 6. Cartoon of generic ELI protocol for isolation of vaccineplasmids for any pathogen.

FIG. 7. Listeria monocytogenes titers from the spleens of ELI-immunized,Listeria monocytogenes-infected mice. 5-6 week old Balb/C female micewere immunized 3 times with 10 mg of Listeria library 1.1 or combinedLis lib 2.1+2.2. Lis Lib are libraries created by ligation of Listeriamonocytogenes genomic Mbol fragments cloned into CMV-GHF1&3 andCMV-GHF-2 as for MP. control (unimmunized) and EKI-immunized mice werechallenged with 10⁵ listeria by i.v. injection and listeria CFUs werecounted from spleen homogenates 3 days later.

FIG. 8. Cartoon showing scheme for sibbing procedure for severalindependent protective plasmids.

FIG. 9. Cartoon showing scheme for sibbing procedure to determinecooperative effects or additive threshold effects.

FIG. 10. Cartoon of the methodology of ELI against Mycoplasma pulmonis(MP). Libraries MP1.1 and MP2.3 consist of two different 3000transformant sibs created by insertion of MP MboI fragments into frame 1of CMV-GH-F1&3 and frame 2 of CMV-GH-F2, respectively. Balb/C mice wereimmunized as indicated using either MP1.1 or MP2.3 by gene gun deliveryand the mice were subsequently challenged with MP and their resistancewas assessed by titering MP from the lungs and by histochemical stainingof lung sections as described in (Lai, et al, 1991). A set of mice withlower MP titers or histopathology indicated that one or more plasmids inthe library confer resistance to MP by genetic immunization.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The invention includes a novel expression library immunization (ELI)method applicable to virtually any pathogen and that requires noknowledge of the biological properties of the pathogen. The methodoperates on the assumption, generally accepted by those skilled in theart, that all the potential antigenic determinants of any pathogen areencoded in its genome. The inventors have now devised methods ofidentifying vaccines using a genomic expression library representing allof the antigenic determinants (except for those resulting frommodification of the protein) of a pathogen. The method has theadvantages of "gene immunization" by eliminating potential for infectionwhile also providing for the first time a general and effective methodof immunizing and identifying a vaccine without having to characterizethe pathogen against which the vaccine is desired.

The preparation of an expression library is performed using thetechniques and methods familiar to the molecular biologist. Thepathogen's genome, whether bacterial, yeast, mold, fungal, algal,protozoan, viral, may or may not be known and may even have been cloned.Thus one obtains DNA (or cDNA), representing substantially the entiregenome of the pathogen. The DNA is broken up, by physical fragmentationor restriction endonuclease, into segments of some length so as toprovide a library of about 10⁻¹ (×genome size) members. Of course forcloned DNA in YACs, a sufficient library may be available. In cases, asfor certain viruses, where the genome is RNA, the RNA may be used toprepare a DNA library. Alternatively, mRNA may be used to generatelibraries; for example from human or animal tumor cells.

The in vivo effectiveness of the novel immunization has beendemonstrated with genomes differing in size by approximately four-fold.The inventors have demonstrated ELI protection against Mycoplasmapulmonis with a genome of ˜1×10⁶ bp and have also shown protectionagainst Listeria monocytogenes, a pathogen whose genome is approximately4×10⁶ bp. These pathogens represent two different classes of pathogens,Mollicutes and Gram-positive bacilli respectively and serve todemonstrate the broad applicability of the method.

One expects the disclosed techniques and methods to apply not only tothe Listeria and Mycoplasma genera but also to the broader classes ofpathogens including Mollicutes and Gram-positive bacilli. Numerousspecies comprise the genera of these classes, including the unusualasporogenous aerobic bacilli represented by Rothia, kurthia andOerskovia. Most pathogens have genomes the same or smaller size thanListeria or Mycoplasma and the method will also apply to larger genomesand will be suitable for developing and identifying vaccines from broadcategories of human and non-human pathogens, shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        viruses        genome of ˜10.sup.3 to 10.sup.5 bp                       mycoplasma     genome of ˜10.sup.6 bp                                   bacteria       genome of ˜2 × 10.sup.6 to 9 × 10.sup.6                     bp                                                             Fungi          genome of ˜2 × 10.sup.7 bp                         Algae          genome of ˜5 × 10.sup.7 bp                         Protozoa       genome of ˜5 × 10.sup.7 bp                         Molds          genome of ˜5 × 10.sup.7 bp to 9 ×                           10.sup.7 bp                                                    cDNA library (any                                                                            ˜10.sup.3 to 10.sup.6 bp                                 pathogen or cancer)                                                           mitochondrial genome                                                                         ˜10.sup.4 to 10.sup.5 bp                                 ______________________________________                                    

1. Other Methods of Inoculation

Introducing an expression library into a subject may be performed inseveral ways; including by gene gun. The gene gun technique used for ELIis thought to be ˜1000 to 10,000-fold more efficient than injection ofnaked DNA (Fynan, et al (1993). Others, e.g. Ulmer, et al (1993) haveindicated that genetic immunization by direct DNA injection may beperformed with similar efficiency as the gene gun. The inventors havefound that gene administration by either method produces qualitativelyidentical immunization. The expression library may also be introduced bymethods other than genetic immunization. The bacteria bearing thelibrary can be directly inoculated into the host or the library and putinto an infectious agent, such as adenovirus. Once the protectingpathogen gene(s) has been isolated the actual vaccine can be by geneticimmunization.

Of course, in light of the new technology on DNA vaccination, it will beunderstood that virtually all such vaccination regimens will beappropriate for use with DNA vectors and constructs, as described byUlmer et al. (1993), Tang et al. (1992), Cox et al. (1993), Fynan et al.(1993), Wang et al. (1993) and Whitton et al. (1993), each incorporatedherein by reference. In addition to parenteral routes of DNAinoculation, including intramuscular and intravenous injections, mucosalvaccination is also contemplated, as may be achieved by administeringdrops of DNA compositions to the nares or trachea. It is particularlycontemplated that a gene-gun could be used to deliver an effectivelyimmunizing amount of DNA to the epidermis (Fynan et al., 1993).

2. ELISAs

ELISAs may be used in conjunction with the invention. In an ELISA assay,proteins or peptides incorporating pathogen antigen sequences areimmobilized onto a selected surface, preferably a surface exhibiting aprotein affinity such as the wells of a polystyrene microtiter plate.After washing to remove incompletely adsorbed material, it is desirableto bind or coat the assay plate wells with a nonspecific protein that isknown to be antigenically neutral with regard to the test antisera suchas bovine serum albumin (BSA), casein or solutions of milk powder. Thisallows for blocking of nonspecific adsorption sites on the immobilizingsurface and thus reduces the background caused by nonspecific binding ofantisera onto the surface.

After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween®. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor from 2 to 4 hours, at temperatures preferably on the order of about25° to about 27° C. Following incubation, the antisera-contacted surfaceis washed so as to remove non-immunocomplexed material. A preferredwashing procedure includes washing with a solution such as PBS/Tweene®,or borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound antigen, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for the first. To provide adetecting means, the second antibody will preferably have an associatedenzyme that will generate a color development upon incubating with anappropriate chromogenic substrate. Thus, for example, one will desire tocontact and incubate the antisera-bound surface with a urease orperoxidase-conjugated anti-human IgG for a period of time and underconditions which favor the development of immunocomplex formation (e.g.,incubation for 2 hours at room temperature in a PBS-containing solutionsuch as PBS-Tween®).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS]and H₂ O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generation, e.g.,using a visible spectra spectrophotometer.

3. Epitopic Core Sequences

The present invention is also directed to protein or peptidecompositions, free from total cells and other peptides, which comprise apurified protein or peptide which incorporates an epitope that isimmunologically cross-reactive with one or more pathogen antibodies, forexample those polypeptides that comprise the epitopic regions ofmycoplasma or Listeria genome.

As used herein, the term "incorporating an epitope(s) that isimmunologically cross-reactive with one or more pathogen antibodies" isintended to refer to a peptide or protein antigen which includes aprimary, secondary or tertiary structure similar to an epitope locatedwithin any of a number of pathogen polypeptides encoded by the pathogenDNA or RNA. The level of similarity will generally be to such a degreethat monoclonal or polyclonal antibodies directed against the suchpolypeptides will also bind to, react with, or otherwise recognize, thecross-reactive peptide or protein antigen. Various immunoassay methodsmay be employed in conjunction with such antibodies, such as, forexample, Western blotting, ELISA, RIA, and the like, all of which areknown to those of skill in the art.

The identification of pathogen epitopes, and/or their functionalequivalents, suitable for use in vaccines is part of the presentinvention. Once isolated and identified, one may readily obtainfunctional equivalents. For example, one may employ the methods of Hopp,as taught in U.S. Pat. No. 4,554,101, incorporated herein by reference,which teaches the identification and preparation of epitopes from aminoacid sequences on the basis of hydrophilicity. The methods described inseveral other papers, and software programs based thereon, can also beused to identify epitopic core sequences (see, for example, Jameson andWolf, 1988; Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acidsequence of these "epitopic core sequences" may then be readilyincorporated into peptides, either through the application of peptidesynthesis or recombinant technology.

Preferred peptides for use in accordance with the present invention willdepend on the particular peptides identified from sib libraryimmunization, as disclosed herein. Relatively short peptides, such asthose prepared from 8 to 30 or so amino acids may provide advantages incertain circumstances, for example, in the preparation of some vaccinesor in immunologic detection assays. Exemplary advantages include theease of preparation and purification, the relatively low cost andimproved reproducibility of production, and advantageousbiodistribution.

It is proposed that particular advantages of the present invention maybe realized through the preparation of synthetic peptides which includemodified and/or extended epitopic/immunogenic core sequences whichresult in a "universal" epitopic peptide directed to pathogen-specificpeptide sequences. These epitopic core sequences are identified hereinin particular aspects as hydrophilic regions of the pathogen antigens.It is proposed that these regions represent those which are most likelyto promote T-cell or B-cell stimulation or humoral response, and, hence,elicit specific antibody production.

An epitopic core sequence, as used herein, is a relatively short stretchof amino acids that is "complementary" to, and therefore will bind,antigen binding sites on transferrin-binding protein antibodies.Additionally or alternatively, an epitopic core sequence is one thatwill elicit antibodies that are cross-reactive with antibodies directedagainst the peptide compositions of the present invention. It will beunderstood that in the context of the present disclosure, the term"complementary" refers to amino acids or peptides that exhibit anattractive force towards each other. Thus, certain epitope coresequences of the present invention may be operationally defined in termsof their ability to compete with or perhaps displace the binding of thedesired protein antigen with the corresponding protein-directedantisera.

In general, the size of the polypeptide antigen is not believed to beparticularly crucial, so long as it is at least large enough to carrythe identified core sequence or sequences. The smallest useful coresequence anticipated by the present disclosure would generally be on theorder of about 8 amino acids in length, with sequences on the order ofup to several tens of amino acids, depending on the size of the DNAfound to in the protective plasmid identified by using the expressionlibrary immunization method. Thus, this size will generally correspondto the smallest peptide antigens prepared in accordance with theinvention. However, the size of the antigen may be relatively large, forexample up to several hundred or more where desired, so long as itcontains a basic epitopic core sequence.

The identification of epitopic core sequences is known to those of skillin the art, for example, as described in U.S. Pat. No. 4,554,101,incorporated herein by reference, which teaches the identification andpreparation of epitopes from amino acid sequences on the basis ofhydrophilicity. Moreover, numerous computer programs are available foruse in predicting antigenic portions of proteins (see e.g., Jameson &Wolf, 1988; Wolf et al., 1988). Computerized peptide sequence analysisprograms (e.g., DNAStar Software, DNAStar, Inc., Madison, Wis.) may alsobe useful in designing synthetic peptides in accordance with the presentdisclosure.

Syntheses of epitopic sequences, or peptides which include an antigenicepitope within their sequence, are readily achieved using conventionalsynthetic techniques such as the solid phase method (e.g., through theuse of commercially available peptide synthesizer such as an AppliedBiosystems Model 430A Peptide Synthesizer). Peptide antigens synthesizedin this manner may then be aliquotted in predetermined amounts andstored in conventional manners, such as in aqueous solutions or, evenmore preferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may bereadily stored in aqueous solutions for fairly long periods of time ifdesired, e.g., up to six months or more, in virtually any aqueoussolution without appreciable degradation or loss of antigenic activity.However, where extended aqueous storage is contemplated it willgenerally be desirable to include agents including buffers such as Trisor phosphate buffers to maintain a pH of about 7.0 to about 7.5.Moreover, it may be desirable to include agents which will inhibitmicrobial growth, such as sodium azide or Merthiolate. For extendedstorage in an aqueous state it will be desirable to store the solutionsat 4° C., or more preferably, frozen. Of course, where the peptides arestored in a lyophilized or powdered state, they may be stored virtuallyindefinitely, e.g., in metered aliquots that may be rehydrated with apredetermined amount of water (preferably distilled) or buffer prior touse.

4. Immunoprecipitation

The antibodies of the present invention are particularly useful for theisolation of antigens by immunoprecipitation. Immunoprecipitationinvolves the separation of the target antigen component from a complexmixture, and is used to discriminate or isolate minute amounts ofprotein. For the isolation of membrane proteins cells must besolubilized into detergent micelles. Nonionic salts are preferred, sinceother agents such as bile salts, precipitate at acid pH or in thepresence of bivalent cations.

In an alternative embodiment the antibodies of the present invention areuseful for the close juxtaposition of two antigens. This is particularlyuseful for increasing the localized concentration of antigens, e.g.enzyme-substrate pairs.

5. Western Blots

The compositions of the present invention may find use in immunoblot orwestern blot analysis. The anti-peptide antibodies may be used ashigh-affinity primary reagents for the identification of proteinsimmobilized onto a solid support matrix, such as nitrocellulose, nylonor combinations thereof. In conjunction with immunoprecipitation,followed by gel electrophoresis, these may be used as a single stepreagent for use in detecting antigens against which secondary reagentsused in the detection of the antigen cause an adverse background. Thisis especially useful when the antigens studied are immunoglobulins(precluding the use of immunoglobulins binding bacterial cell wallcomponents), the antigens studied cross-react with the detecting agent,or they migrate at the same relative molecular weight as across-reacting signal.

Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the peptide moiety areconsidered to be of particular use in this regard.

6. Vaccines

The present invention contemplates vaccines for use in both active andpassive immunization embodiments. Immunogenic compositions, proposed tobe suitable for use as a vaccine, may be prepared most readily directlyfrom immunogenic pathogen sib expression libraries in the mannerdisclosed herein. Preferably the antigenic material is extensivelydialyzed to remove undesired small molecular weight molecules and/orlyophilized for more ready formulation into a desired vehicle. Vaccinesmay be polypeptide or DNA compositions. DNA compositions are preferablycloned sib expression libraries, obtained from the fragmented genome ofa pathogen.

The inventors have demonstrated that one may generate an immune responsein an animal by administering to the animal, or human subject, apharmaceutically acceptable composition comprising an immunologicallyeffective amount of a cloned expression nucleic acid library. Thestimulation of specific antibodies and CTL (cytotoxic T lymphocyte)responses upon administering to an animal a nucleic molecule is now wellknown in the art, as evidenced by articles such as Tang et al. (1992);Cox et al. (1993;) Fynan et al. (1993); Ulmer et al. (1993); Wang et al.(1993) and Whitton et al. (1993); each incorporated herein by reference.

This technology, often referred to as genetic immunization, isparticularly suitable to protect against bacterial infections and isexpected to be equally protective against viral infections. Indeed,immunization with DNA has been successfully employed to protect animalsfrom challenge with influenza A (Ulmer et al., 1993). Therefore, the useof the expression library compositions of the present inventionemploying techniques similar to those described by Ulmer et al. (1993,incorporated herein by reference), is considered to be particularlyuseful as a vaccination regimen.

The expression library DNA segments can be used in virtually any form,including naked DNA and plasmid DNA, and may be administered to theanimal in a variety of ways, including parenteral, mucosal and gene-guninoculations, as described, for example, by Fynan et al. (1993) and Tanget al (1992).

The inventors have used expression plasmids for immunization; however,it is contemplated that the DNA segments themselves as immunizing agentsto vaccinate against infection and disease. The technology for using DNAsegments as vaccines has recently been developed and is generally termed"Genetic Immunization" or "DNA Vaccination" (Cohen, 1993). It is nowknown that cells can take up naked DNA and express the peptides encodedon their surface, thus stimulating an effective immune response, whichincludes the generation of cytotoxic T lymphocytes (killer T cells).

The utilization of this technology, and variations thereof, such asthose described by Tang et al. (1992), Ulmer et al. (1993); Cox et al.(1993), Fynan et al. (1993), Wang et al. (1993) and Whitton et al.(1993), each incorporated herein by reference, is particularly suitableas it has already been shown to be successful against a form ofinfluenza virus, the type of pathogen also targeted by the presentinvention. It is contemplated that virtually any type of vector,including naked DNA in the form of a plasmid, could be employed togenerate an immune response in conjunction with a wide variety ofimmunization protocols, including parenteral, mucosal and gene-guninoculations (Tang et al, 1992; Fynan et al., 1993).

The preparation of vaccines which contain peptide sequences, determinedfrom the DNA of plasmids identified as protective against pathogenchallenge, as active ingredients is generally well understood in theart, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231;4,599,230; 4,596,792; and 4.578,770, all incorporated herein byreference. Typically, such vaccines are prepared as injectables, eitheras liquid solutions or suspensions or solid forms suitable for solutionin, or suspension in, liquid prior to injection. The preparation mayalso be emulsified. The active immunogenic ingredient is often mixedwith excipients which are pharmaceutically acceptable and compatiblewith the active ingredient. Suitable excipients are, for example, water,saline, dextrose, glycerol, ethanol, or the like and combinationsthereof. In addition, if desired, the vaccine may contain minor amountsof auxiliary substances such as wetting or emulsifying agents, pHbuffering agents, or adjuvants which enhance the effectiveness of thevaccines.

Vaccines may be conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkalene glycols or triglycerides: such suppositories maybe formed from mixtures containing the active ingredient in the range ofabout 0.5% to about 10%, preferably about 1 to about 2%. Oralformulations include such normally employed excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain about 10to about 95% of active ingredient, preferably about 25 to about 70%.

The pathogen peptides of the present invention may be formulated intothe vaccine as neutral or salt forms. Pharmaceutically-acceptable salts,include the acid addition salts (formed with the free amino groups ofthe peptide) and those which are formed with inorganic acids such as,for example, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed with thefree carboxyl groups may also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g., the capacity of the individual's immunesystem to synthesize antibodies, and the degree of protection desired.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges areof the order of several hundred micrograms active ingredient pervaccination. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These arebelieved to include gene gun inoculation of the DNA encoding the antigenpeptide(s), phage transfection of the DNA, oral application on a solidphysiologically acceptable base or in a physiologically acceptabledispersion, parenterally, by injection or the like. The dosage of thevaccine will depend on the route of administration and will varyaccording to the size of the host.

Various methods of achieving adjuvant effect for the vaccine includesuse of agents such as aluminum hydroxide or phosphate (alum), commonlyused as about 0.05 to about 0.1% solution in phosphate buffered saline,admixture with synthetic polymers of sugars (Carbopol®) used as an about0.25% solution, aggregation of the protein in the vaccine by heattreatment with temperatures ranging between about 70° to about 101° C.for a 30-second to 2-minute period, respectively. Aggregation byreactivating with pepsin treated (Fab) antibodies to albumin, mixturewith bacterial cells such as C. parvum or endotoxins orlipopolysaccharide components of Gram-negative bacteria, emulsion inphysiologically acceptable oil vehicles such as mannide mono-oleate(Aracel A) or emulsion with a 20% solution of a perfiluorocarbon(Fluosol-DA®) used as a block substitute may also be employed.

In many instances, it will be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and preferably one or more, usually at leastabout three vaccinations. The vaccinations will normally be at from twoto twelve week intervals, more usually from three to five weekintervals. Periodic boosters at intervals of 1-5 years, usually threeyears, will be desirable to maintain protective levels of theantibodies. The course of the immunization may be followed by assays forantibodies for the supernatant antigens. The assays may be performed bylabeling with conventional labels, such as radionuclides, enzymes,fluorescents, and the like. These techniques are well known and may befound in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932;4,174,384 and 3,949,064, as illustrative of these types of assays.

7. DNA Segments Encoding Novel Peptides

The present invention also concerns DNA segments, that can be isolatedfrom virtually any non-mammalian pathogen source, that are free fromtotal genomic DNA and that encode the novel peptides disclosed herein.DNA segments encoding these peptide species may prove to encodeproteins, polypeptides, subunits, functional domains, and the like ofpathogen or other non-related gene products. In addition these DNAsegments may be synthesized entirely in vitro using methods that arewell-known to those of skill in the art.

As used herein, the term "DNA segment" refers to a DNA molecule that hasbeen isolated free of total genomic DNA of a particular species.Therefore, a DNA segment encoding a pathogen peptide refers to a DNAsegment that contains these peptide coding sequences yet is isolatedaway from, or purified free from, total genomic DNA of the species fromwhich the DNA segment is obtained. Included within the term "DNAsegment", are DNA segments and smaller fragments of such segments, andalso recombinant vectors, including, for example, plasmids, cosmids,phagemids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified pathogenpeptide-encoding gene refers to a DNA segment which may include inaddition to peptide encoding sequences, certain other elements such as,regulatory sequences, isolated substantially away from other naturallyoccurring genes or protein-encoding sequences. In this respect, the term"gene" is used for simplicity to refer to a functional protein-,polypeptide- or peptide-encoding unit. As will be understood by those inthe art, this functional term includes both genomic sequences, cDNAsequences and smaller engineered gene segments that express, or may beadapted to express, proteins, polypeptides or peptides.

"Isolated substantially away from other coding sequences" means that thegene of interest, in this case, a gene encoding pathogen epitopes ofpolypeptides forms the significant part of the coding region of the DNAsegment, and that the DNA segment does not contain large portions ofnaturally-occurring coding DNA, such as large chromosomal fragments orother functional genes or cDNA coding regions. Of course, this refers tothe DNA segment as originally isolated, and does not exclude genes orcoding regions later added to the segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences that encode pathogenantigenic species that includes within its amino acid sequence an aminoacid sequence that essentially include one or more amino acid sequencesof epitopic regions of the pathogen polypeptide.

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 50¢ or 3¢ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5¢ or 3¢ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, nucleic acid fragments may be prepared thatinclude a short contiguous stretch encoding any of the immunogenicpolypeptide sequences identified by the methods herein disclosed, orthat are identical to or complementary to DNA sequences which encode anyof these peptides.

It will be readily understood that "intermediate lengths", in thesecontexts, means any length between the quoted ranges, such as 14, 15,16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51,52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.;including all integers through the 200-500; 500-1,000; 1,000-2,000;2,000-3,000; 3,000-5,000; and up to and including sequences of about10,000 nucleotides and the like.

It will also be understood that this invention is not limited to theparticular nucleic acid sequences which encode peptides of Mycoplasma orListeria or only to pathogen DNA encoding only epitopic regions.Recombinant vectors and isolated DNA segments may therefore variouslyinclude the peptide-coding regions themselves, coding regions bearingselected alterations or modifications in the basic coding region, orthey may encode larger polypeptides that nevertheless include thesepeptide-coding regions or may encode biologically functional equivalentproteins or peptides that have variant amino acids sequences.

The DNA segments of the present invention encompassbiologically-functional equivalent peptides. Such sequences may arise asa consequence of codon redundancy and functional equivalency that areknown to occur naturally within nucleic acid sequences and the proteinsthus encoded. Alternatively, functionally-equivalent proteins orpeptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein or to test mutants inorder to examine activity at the molecular level.

If desired, one may also prepare fusion proteins and peptides, e.g.,where the peptide-coding regions are aligned within the same expressionunit with other proteins or peptides having desired functions, such asfor purification or immunodetection purposes (e.g., proteins that may bepurified by affinity chromatography and enzyme label coding regions,respectively).

Recombinant vectors form further aspects of the present invention.Particularly useful vectors are contemplated to be those vectors inwhich the coding portion of the DNA segment, whether encoding afull-length protein or smaller peptide, is positioned under the controlof a promoter. The promoter may be in the form of the promoter that isnaturally associated with a gene encoding peptides of the presentinvention, as may be obtained by isolating the 5¢ non-coding sequenceslocated upstream of the coding segment or exon, for example, usingrecombinant cloning and/or PCRO technology, in connection with thecompositions disclosed herein.

In other embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is intended to refer to a promoter that is notnormally associated with a DNA segment encoding a pathogen peptide inits natural environment. Such promoters may include promoters normallyassociated with other genes, and/or promoters isolated from anybacterial, viral, eukaryotic, or mammalian cell. Naturally, it will beimportant to employ a promoter that effectively directs the expressionof the DNA segment in the cell type, organism, or animal, chosen forexpression. The use of promoter and cell type combinations for proteinexpression is generally known to those of skill in the art of molecularbiology, for example, see Sambrook et al., 1989. The promoters employedmay be constitutive, or inducible, and can be used under the appropriateconditions to direct high level expression of the introduced DNAsegment. An appropriate promoter for use in high-level expression is thecytomegalovirus promoter (Pharmacia LKB Biotechnology), although one isnot limited to use of this promoter.

In connection with expression embodiments to prepare recombinantproteins and peptides, it is contemplated that longer DNA segments willmost often be used, with DNA segments encoding the entire peptidesequence being most preferred. However, it will be appreciated that theuse of shorter DNA segments to direct the expression of pathogenepitopic core regions, such as may be used to generate anti-peptideantibodies, also falls within the scope of the invention. DNA segmentsthat encode peptide antigens from about 8 to about 50 amino acids inlength, from about 8 to about 30 amino acids in length, or even fromabout 8 to about 20 amino acids in length are contemplated to beparticularly useful.

In addition to their use in directing the expression of the pathogenpeptides of the present invention, the nucleic acid sequencescontemplated herein also have a variety of other uses. For example, theyalso have utility as probes or primers in nucleic acid hybridizationembodiments. As such, it is contemplated that nucleic acid segments thatcomprise a sequence region that consists of at least a 14 nucleotidelong contiguous sequence that has the same sequence as, or iscomplementary to, a 14 nucleotide long contiguous DNA segment will findparticular utility. Longer contiguous identical or complementarysequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000(including all intermediate lengths) and even up to full lengthsequences will also be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize topeptide-encoding sequences will enable them to be of use in detectingthe presence of complementary sequences in a given sample. However,other uses are envisioned, including the use of the sequence informationfor the preparation of mutant species primers, or primers for use inpreparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguousnucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200nucleotides or so, identical or complementary to DNA sequences of any ofthe DNAs disclosed, are contemplated as hybridization probes for use in,e.g., Southern and Northern blotting. Smaller fragments will generallyfind use in hybridization embodiments, wherein the length of thecontiguous complementary region may be varied, such as between about10-14 and about 100 nucleotides, but larger contiguous complementaritystretches may be used, according to the length complementary sequencesone wishes to detect.

The use of a hybridization probe of about 10-14 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 10 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 20 contiguous nucleotides,or even longer where desired.

Of course, fragments may also be obtained by other techniques such as,e.g., by mechanical shearing or by restriction enzyme digestion. Smallnucleic acid segments or fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCRO technology of U.S. Pat. Nos. 4,683,195 and4,683,202 (each incorporated herein by reference), by introducingselected sequences into recombinant vectors for recombinant production,and by other recombinant DNA techniques generally known to those ofskill in the art of molecular biology.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNA fragments. Depending on the application envisioned, onewill desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of probe towards target sequence. Forapplications requiring high selectivity, one will typically desire toemploy relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.15 M NaCl at temperatures of 50°C. to 70° C. Such selective conditions tolerate little, if any, mismatchbetween the probe and the template or target strand. Detection of DNAsegments via hybridization is well-known to those of skill in the art,and the teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 (eachincorporated herein by reference) are exemplary of the methods ofhybridization analyses. Teachings such as those found in the texts ofMaloy et al., 1993; Segal 1976; Prokop, 1991; and Kuby, 1991, areparticularly relevant.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate pathogenpeptide-encoding sequences from related species, functional equivalents,or the like, less stringent hybridization conditions will typically beneeded in order to allow formation of the heteroduplex. In thesecircumstances, one may desire to employ conditions such as about 0.15 Mto about 0.9 M salt, at temperatures ranging from about 20° C. to about55° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In preferred embodiments, onewill likely desire to employ a fluorescent label or an enzyme tag, suchas urease, alkaline phosphatase or peroxidase, instead of radioactive orother environmental undesirable reagents. In the case of enzyme tags,calorimetric indicator substrates are known that can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C content, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantitated, by means of the label.

8. Biological Functional Equivalents

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thefollowing codon table:

                  TABLE 1                                                         ______________________________________                                        Amino Acids                                                                            Codons                                                               ______________________________________                                        Alanine  Ala    A      GCA  GCC  GCG  GCU                                     Cysteine Cys    C      UGC  UGU                                               Aspartic acid                                                                          Asp    D      GAC  GAU                                               Glutamic acid                                                                          Glu    E      GAA  GAG                                               Phenylalanine                                                                          Phe    F      UUC  UUU                                               Glycine  Gly    G      GGA  GGC  GGG  GGU                                     Histidine                                                                              His    H      CAC  CAU                                               Isoleucine                                                                             Ile    I      AUA  AUC  AUU                                          Lysine   Lys    K      AAA  AAG                                               Leucine  Leu    L      UUA  UUG  CUA  CUC  CUG  CUU                           Methionine                                                                             Met    M      AUG                                                    Asparagine                                                                             Asn    N      AAC  AAU                                               Proline  Pro    P      CCA  CCC  CCG  CCU                                     Glutamine                                                                              Gln    Q      CAA  CAG                                               Arginine Arg    R      AGA  AGG  CGA  CGC  CGG  CGU                           Serine   Ser    S      AGC  AGU  UCA  UCC  UCG  UCU                           Threonine                                                                              Thr    T      ACA  ACC  ACG  ACU                                     Valine   Val    V      GUA  GUC  GUG  GUU                                     Tryptophan                                                                             Trp    W      UGG                                                    Tyrosine Tyr    Y      UAC  UAU                                               ______________________________________                                    

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8);tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2);glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5);lysine (-3.9); and arginine (-4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8);tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

9. Site-Specific Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, for example, incorporating one or more of the foregoingconsiderations, by introducing one or more nucleotide sequence changesinto the DNA. Site-specific mutagenesis allows the production of mutantsthrough the use of specific oligonucleotide sequences which encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art. Double stranded plasmids are alsoroutinely employed in site directed mutagenesis which eliminates thestep of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.

10. Monoclonal Antibody Generation

Means for preparing and characterizing antibodies are well known in theart (See, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference).

The methods for generating monoclonal antibodies (mAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogenic composition in accordance with the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified epitopic protein, polypeptide or peptide. Theimmunizing composition is administered in a manner effective tostimulate antibody producing cells. Rodents such as mice and rats arepreferred animals, however, the use of rabbit, sheep frog cells is alsopossible. The use of rats may provide certain advantages (Goding, 1986,pp. 60-61), but mice are preferred, with the BALB/c mouse being mostpreferred as this is most routinely used and generally gives a higherpercentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5'10⁷ to 2'10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp.65-66, 1986; Campbell, pp.75-83, 1984).For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described by Kohler andMilstein (1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, by Gefter et al., (1977). The use of electricallyinduced fusion methods is also appropriate (Goding pp. 71-74, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1'10⁻⁶ to 1'10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immuno-bindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Materials and Methods

Vector Constructs. CMV-GH was constructed by insertion of the genomichGH sequence from CMV-GH-ori (Barry et al, 1994) as a BamHI fragmentinto the BglII site of CMV5, a derivative of CMV1 (Andersson et al,1989)]. CMV-GH-F1&3 was constructed by knocking out the BamHI in CMV-GHand inserting the annealed, kinased oligonucleotides,GATCTTGGATCCTAAGTAAGTA (SEQ ID NO:1) and AGCTTACTTACTTAGGATCCAA (SEQ IDNO:2), into BglII-HindIII-digested CMV-GH. CMV-GH-F2 was constructedsimilarly by inserting the oligos, GATCGGATCCTAAGTAAGTA (SEQ ID NO:3)and AGCTTACTTACTTAGGATCC (SEQ ID NO:4) in BglII-HindIII-digested CMV-GH.Mycoplasma pulmonis strain CT was grown in Chalquest media for isolationof genomic DNA. Listeria monocytogenes was grown in LB. Genomic DNA wasisolated from each as described Ausubel, 1992. Each genomic DNA waspartially digested with MboI to an approximate mean fragment size of 0.5kilobase pairs. CMV-GH-F1&3 and CMV-GH-F2 were digested with BamHI orBglII and dephosphorylated with shrimp alkaline phosphatase. Each vectorwas ligated with a 5-fold excess of genomic MboI fragments andelectroporated into TG1 bacteria. Transformant number was estimated byplating serial dilutions onto YT-ampicillin plates and approximately3000 transformants were grown overnight in LB-ampicillin and frozen. 5ml of this overnight grow up was used to inoculate a 500 ml LB-ampculture from which plasmid DNA was prepared using Qiagen plasmidpurification columns.

Animals. Mice were treated in accordance with institutional guidelines.Prior to immunization, the mice were anesthetized with 0.5 ml of avertini.p. and their ears depilated with NairO. Up to 2.5 mg of plasmid DNAwas loaded on 0.5 mg of 1-3 mm gold microparticles for each inoculum.The total amount of DNA to be delivered was delivered in 4 inoculumsinto both sides of both ears using the hand-held biolistic gene gundescribed in Sanford, et al, 1991.

Measurement of Delayed-type Hypersensitivity (DTH). DTH was evaluated byinjecting PBS into the right rear footpad and PBS containing 50 mgsonicated MP cell protein into the left rear footpad. A dial gaugecaliper was used to measure the change in footpad thickness induced 24 hafter injection. Three readings were measured and averaged.

Macrophage Migration Inhibition (MMI). MMI was evaluated by filling aglass capillary tube with 100 ml of spleen cell suspension (1×10⁸cells/ml) from each mouse and placing it horizontally in a well of a 24well plate immersed in RPMI media in the absence or presence of 50 g/mlsonicated MP protein. After 24 h, the area of cell migration out of thetube was measured by digital imaging. The area of migrated cells fromcontrol animal was averaged. Less migration was indicative of release ofMIF from T-cells previously activated against MP antigens byimmunization. Release of MIF in this assay results in reduced area ofmigrated cells from the capillary.

EXAMPLE 1

This example illustrates that a diverse library of plasmids can beinoculated and still produce an immune response to each encoded antigen.Theoretically a library including all the genome could be inoculated,exposing the host to all the pathogen's proteins. However, inoculationwith 1 mg of a library of 10,000, for example, would result in deliveryof only 0.1 ng of each individual plasmid. This demonstration wasimportant for addressing the feasibility of ELI. Previous work had shownthat inoculation of 1 mg of DNA encoding hGH produced only approximately0.1 ng of protein. Typical procedures for immunization utilize 10-100 mgof protein.

Determination of Amount Required for Immune Response

Mice were inoculated with various amounts of DNA encoding human growthhormone (hGH) and tested for antibodies against hGH (FIG. 2).Considering the need for efficiency, the gene gun was used rather thanneedle injection into muscle or dermis, since the gene gun appears torequire less DNA for a given response (Fynan et al. 1993).

Mouse sera was recovered by tail vein bleed 10 days after geneinoculation. hGH antibodies were measured by a modified ELISA protocolwhich makes use of the highly sensitive, wide range, luminescentb-galactosidase assay (GalactolightO-Tropix) able to detect fg to ng ofb-galactosidase activity.

100 ng of hGH protein in 100 ml PBS (137 mM NaCl, 2.7 mM KCl, 8.2 mM Na₂HPO₄, 1.5 mM KH₂ PO₄, pH 7.4) was coated into each well of a 96 wellplate ELISA plate for 2 h at room temp. The wells were blocked by theaddition of 300 ml of 5% dried milk in TBST (150 mM NaCl, 10 mM Tris, pH8, 0.1% Tween) for 1 h. The wells were washed 3 times with TBST and 200ml of a 1/250 dilution of mouse sera was added for 2 h at room temp.This solution was removed, the wells were washed 6 times with TBST and200 ml of a 1/1000 dilution of goat anti-mouse IgG-b-galactosidaseconjugate in TBST was added for 1 h. The wells were washed 5 times inTBST and once with PBS. 200 ml of complete Galacto-light reaction buffer(1/100 dilution of galacton concentrate in reaction buffer) was added toeach well and incubated for 30 to 60 min at room temp. This solution wasthen transferred to luminometer cuvettes, 300 ml of Galactolightaccelerator was added and luminescence was measured 5 sec later byintegration for 10 sec on a Berthold BIOLUMAT 9500C luminometer.Relative antibody titers are expressed in b-galactosidase lumens (FIG.2).

Surprisingly, antibodies were detectable following inoculation of aslittle as 1 ng of hGH DNA, consistent with previous reports (Eisenbraun,et al, 1993). This indicated that libraries of at least 10³ -10⁴ memberswere possible with 1 mg of DNA.

EXAMPLE 2

Expression library immunization in mice was tested using the pathogenMycoplasma pulmonis (MP), a wall-less bacteria. MP has a relativelysmall genome of ˜10⁶ base pairs (Neimark and Lange, 1990). MP is anextracellular pathogen that colonizes the lungs and other tissues(Lindsey et al, 1978). This bacterium was considered to pose a challengefor creating a representative library because it has an unusual codonusage (i.e. the tryptophan codon of MP is a stop in mammalian cells(Yamao et al, 1985) so that it appeared problematical that wider rangeexpression of a library would be possible in mammalian cells.Additionally, MP is an endogenous pathogen in rodents (Cassell, 1982),often causing losses in animal supply colonies, and any effectivevaccine discovered would be a potential benefit to those maintaininglarge rodent colonies.

Mycoplasm vaccination with a Mycoplasm Sib Library

A library was constructed by inserting partially digested MP DNA intothe last exon of the hGH gene under control of the CMV promoter. The hGHgene contains a signal sequence allowing MP antigens to be secreted asfusion proteins. Since the fragments of MP DNA inserted randomly, only1/6 of the ones corresponding to open reading frames would be expectedto be in-frame. To include all possible antigens, MP fragments werefused into 3 different frames of hGH sequence and with stop codons in 3frames at the 3' end (FIG. 3). Nine independent (sib) libraries wereconstructed, each with ˜3000 members. Over 95% of the plasmids boreinserts with a median size of ˜400 bp.

Two of these sib libraries (MP1.1 and MP2.3) were inoculated separatelyinto the skin of the ear of mice, with 10 mg introduced in a total offour inoculation sites. These inoculations contained ˜1 mg of MP DNA,representing the equivalent of ˜1×10⁹ MP genomes--10⁶ -fold more thanintroduced in a normal MP infection. As negative controls, a plasmidencoding hGH alone or a comparable hGH fusion library with DNA fromListeria monocytogenes were inoculated in the same fashion. Sixty daysafter the first inoculation, and ten days after the last inoculation themice were challenged by intranasal introduction of MP. Two weeks later,the mice were tested for MP infection. All of the control mice(non-inoculated and inoculated with hGH or Listeria DNA) had 10⁵ to 10⁷mycoplasma in lung lavages even after the lowest (10³) challenge. Lungsections from these mice showed significant lesions in the lung at thelowest challenge (FIGS. 4A and 4B).

In remarkable contrast, the mice inoculated with either the MP1.1 orMP2.3 library had no culturable mycoplasma even at the highest challengeand showed no evidence of lung lesions (FIG. 5).

Anti-mycoplasma immune responses were characterized by several assays(Table 1). Mice vaccinated with libraries MP1.1 and MP2.3 demonstratedstrong delayed-type hypersensitivity (DTH) to MP proteins, while therewas little or no response in the control animals. Histologicalexamination demonstrated massive mononuclear cell infiltration in the MPlibrary-injected mice but not in control mice (data not shown). TheseDTH responses indicate that T-cells have been activated againstmycoplasma antigens by inoculation of the MP libraries. Similarly,T-cells from mice immunized with MP1.1 or MP2.3 were primed tomycoplasma antigens and released migration inhibition factor (MIF) inmacrophage migration inhibition tests. Mice were immunized as describedin FIG. 6. Anti-hGH and anti-MP antibodies were measured by ELISA fromsera taken 10 days after the second inoculation. 2 mice from each groupwere tested for DTH and MMI 12 days after the last immunization. Controlrefers to un-immunized mice. Results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Immune Responses Induced by ELI Libraries.                                    Anti-hGH     Anti-MP    MP-specific                                                                             MP-specific                                 Antibodies.sup.1                                                                           Antibodies.sup.2                                                                         DTH (mm).sup.3                                                                          MMI (%).sup.4                               ______________________________________                                        Control                                                                              -         -           2.3 ± 0.4                                                                         0                                         MP 1.1 +         +          16.4 ± 0.4                                                                         71.8                                      MP 2.3 +         +          19.8 ± 0.3                                                                         73.3                                      ______________________________________                                         .sup.1 Antibody levels against hGH protein. (-) designates no antibodies,     (+) indicates levels detectable only at dilutions of 1/250.                   .sup.2 Antibody levels against whole mycoplasma antigens. (-) designates      no antibodies, (+) indicates levels detectable only at dilutions of 1/50.     .sup.3 MPspecific delayedtype hypersensitivity. Measurements indicate the     change in footpad thickness induced by injection of MP antigens in PBS        relative to that of PBS alone (20). Net footpad thickness (×100 mm)     = [(postMP injection minus preMP injection) minus postPBS injection].         .sup.4 MPspecific macrophage migration inhibition. Percent inhibition was     calculated from the formula: (A - B)/A × 100, where A = the area of     macrophage migration in media and B = the area of macrophage migration in     media containing MP antigen (21).                                        

Sera from MP1.1 and MP2.3 mice showed relatively low titers ofantibodies against hGH and mycoplasma proteins (Table 1). Though alllibrary members encode hGH and inoculation of hGH alone induced strongantibody titers, the fusion proteins may be restricted in their abilityto be secreted and produce a humoral response. A similar low titer ofhGH antibodies was observed with a Listeria library.

In another experiment, mice were immunized three times in a 30 dayrather than 60 day regime to determine how rapidly protection could beinitiated. This shortened protocol elicited substantial protection withthe control mice having 10⁴ more culturable pathogen at a given initialinoculum. However, unlike in the first experiment the protection was notas complete. This difference may arise because of the longer period ofimmune response before the challenge.

EXAMPLE 3

A Listeria ELI library was created in the same manner as that formycoplasma.

Expression Library Immunization with Listeria.

Genomic DNA from Listeria monocytogenes was isolated and partiallydigested to an approximate mean fragment size of 0.4 kilobase pairs.These fragments were ligated into the human growth hormone sequence togenerate a library which was sibbed into 21 sub-libraries ofapproximately 3000 transformants each. A larger set of sibs wasconstructed since the genome size of Listeria is approximately 4 timesthat of Mycoplasma.

A total of 10 mg of the indicated 3000 transformant sib(s) or parentplasmid was loaded on 0.5 mg of 1-3 mm gold microparticles and deliveredinto the ears of anesthetized 5-6 week old female Balb/C mice using ahand-held biolistic gene gun (Sanford et al. (1991)). The gene gun wasused to maximize the efficiency of immunization. Mice were immunized 3times over 60 days and challenged 7 days after final immunization withapproximately 1×10⁵ Listeria monocytogenes by intravenous injection intothe tail vein. Three days later, the mice were sacrificed and listeriarecovered from their spleens and counted.

Results are shown in FIG. 7. Inoculation with two of the sib librariesprovided substantial protection on challenge with Listeria monocytogenescompared with controls.

EXAMPLE 4

The in vivo methods for identifying and developing expression libraryvaccines as described in Examples 2 and 3 are equally applicable totesting and sibbing of libraries ex vivo. This example illustrates an exvivo method of identifying a vaccine.

Ex vivo Identification of Sib Library Vaccines

Cells or sera from an animal immunized with a sib are tested forreaction against mammalian cells transfected with the same or anothersib. To test for cellular responses, transfected cells are plated in 96well plates a single or multiple clones per well. Cells from the blood,spleen, lymph nodes, or other sites are added to the wells and eitherCTL activity, proliferation, or cytokine secretion measured. A well inwhich a positive reaction occurs will indicate that the antigen genetransfected into the cell elicited the particular immune response whichwas assayed.

This approach allows particular modes of immune responses to be screenedto avoid known deleterious immune events such as autoimmune damage inChaga's disease (Parham, (1994). A similar approach is used withbacterial, viral, yeast, or other cellular carriers in the absence orpresence of antigen presenting cells such as macrophages. Antibodiesfrom sera or other sites could be tested against purified ELI antigens(e.g. glutathione fusions) or cellular extracts from the above carriersin an antibody capture ELISA. For those antigens that are secreted orlocated on the surface of the carrier, antibodies from the immunizedmouse can capture these antigens in ELISA. Although most screening willpreferably be performed using cells or antibodies from ELI-immunizedanimals, similar ex vivo screening may be performed using reagents fromanimals infected with the legitimate pathogen.

EXAMPLE 5

The inventors expect that MP genomic fragments may be cloned into the afragment of b-galactosidase such that mycoplasma antigens will besynthesized inside the E. coli. Engulfment of the bacteria will resultin presentation of the mycoplasma antigen and immunization.

A similar library may be constructed where the mycoplasma antigens aresecreted from the E. coli into the gut where they will activate IgAimmunity which extends from the gastrointestinal tract into the lungsand nasal passages. These bacterial libraries will be introduced inother locations such as the peritoneum or sub-cutaneously to elicitimmunity in other locations. The bacterial libraries can be introducedin osmotic pumps to avoid infection or as killed preparations.

An alternate bacterial host for the libraries is Listeria monocytogenes,since this bacterium invades macrophages and elicits MHC classI-restricted antigen presentation (Schafer, et al, 1992).

Antigen libraries can also be built onto the coat proteins ofbacteriophages. For example, antigens can be added to the plll and pVIIIproteins of bacteriophage fdTET. An advantage of bacteriophage librariesis that they are expected to have no pathologic effect on the organismunlike living vectors.

Finally, ELI antigens can be created in vitro in E. coli expressionsystems by fusion to glutathione S-transferase or another fusion proteinand inoculated into animals as a soluble protein bolus to elicitantibody responses.

Alternately, the library proteins can be introduced on agarose,paramagnetic, iron, or latex beads which can elicit cytotoxic Tlymphocyte activity following phagocytosis of the beads by macrophages(Kovacsovics-Bankowski, et al, 1993).

EXAMPLE 6

In addition to inoculation of DNA into animals, it is expected that ELIcan be performed using pathogen libraries transfected into syngeneicanimal cells and the cells subsequently introduced into the animal. Oncein the animal, the transfected cells can present library antigens toelicit immune responses. Library antigens may also be cloned intomammalian viruses such as adenovirus or vaccinia and inoculated intoanimals or cells rather than naked DNA.

Other approaches include expressing library antigens in E. coli or otherbacteria (e.g. listeria), yeast, bacteriophages, or other cellularvehicles which can be introduced into the animal to elicit an immuneresponse. Others have demonstrated that E. coli expressing a singlemycoplasma antigen fused to b-galactosidase introduced into the gut ofmice elicits immunity to mycoplasma (Lai, et al, 1994).

A variety of approaches are contemplated for the isolation of individualvaccine genes using ELI. The success of each depends on the number anddegree of cooperative effects between antigen genes. Two types ofprotective genes are expected to be detected in the practice of thedisclosed method in the course of sibbing a library. As an example, afinite set of antigen gene fragments that are independently protectivemay be found for a particular pathogen. For these types of genes, asequential sibbing protocol can be used in which a positive sib isdivided into smaller sibs to be tested in the target organism forprotection (FIG. 8). Reiterative sibbing and testing for protection willeventually isolate individual protective genes from the library. As theproportion of protective gene fragment increases in sequential sibs,protection should increase to some maximal amount. For example, in a3000 transformant sib, only 3 ng of a single independent protectiveclone is delivered to an animal in 10 mg of sib DNA. After sibbing to a100 transformant library now 100 ng of the positive clone would bedelivered in a 10 mg immunization resulting in as much as a 33-foldincrease in protection (up to a certain level). It is expected thatthese types of genes will have additive protective effects when combinedwith other genes of this class.

There may also be antigen genes that require the presence of one or moreother antigen genes to confer resistance. In this situation, sequentialsibbing will eventually result in near total loss of protection in allsibs at a certain level of division. To isolate these cooperative gene,a positive sib is divided into overlapping halves (FIG. 9) and tested inanimals for protection. If only one half sib conferred resistance, thisindicates that two or more cooperative genes are located in the twoquarters and that loss of either totally abrogates protection. Apositive half sib is then broken in alternate patterns to isolate asmaller sib containing the protective plasmids. This process isreiterated until the minimal set of genes is isolated.

Both approaches may be tested in parallel since both classes (andothers) may be observed. Computer controlled sibbing is expected tofacilitate this process since individual DNA isolates can be created atthe beginning of the sibbing process and algorithms can be created tocombine clones in either approach.

EXAMPLE 7

The expression library is constructed to represent at least asubstantial portion of the pathogens genome. Bacterial pathogens havegenomes of approximately 3×10⁶ base pairs. If each fragment is about1×10³ bp, a genome equivalent would be approximately 1.8×10⁴ clonesconsidering only 1/16 would be in frame. To capture all possible codingsequences, the fragments are ligated into vectors that place them intoeach of three forward coding frames, thereby generating a set of threemaster libraries. Three genome equivalents for each master library wouldbe 5.4×10⁴ clones. The master genomic expression library is maintainedas a collection of sib libraries, each containing 1/10 to 1/20 of thetotal. The size of the sib library is dictated by certainconsiderations, including the lowest amount of DNA that can beinoculated and still produce an immune response.

Mycoplasm protection has been achieved with two different libraries of3000 transformants (MP1.1 and MP2.3) containing an unknown number ofprotective antigen gene fragments. These sibs differ in that mycoplasmafragments were cloned into two different coding frames of human growthhormone. It thus appears unlikely that the same antigen fragment isconferring protection in both libraries, indicating that there areminimally 2 independent protective genes or an unknown number ofcooperative protective genes in 6000 total transformants. Since only 1/6of clones should express a real mycoplasma protein fragment, a minimalestimate is 1/500 expressing clones are protective. To estimate themaximal number of protective independent clones, 2 sibs were createdfrom MP2.3 in which each contained 69 transformants (MP2.3.01 andMP2.3.02) and approximately 11 mycoplasma antigen-expressing cloneseach. Inoculation of these sibs into mice resulted in little or noprotection. This suggests that the maximum number of independentprotective clones is less than 1/22 expressing clones. From this it isestimated that there are approximately 10 to 200 independent protectiveantigen genes and an unknown number of cooperative genes in the totalmycoplasma library of 27,000 transformants. A complete sibbed mycoplasmalibrary sibbed to completion will allow an accurate estimate for thisorganism. FIG. 10 is a cartoon summarizing in general the immunizationprotocol.

For viral pathogens, sibbing will be proportionally simplified sincetheir genomes are 10 to 1000-fold smaller than mycoplasma.

The results of the present work indicate that different organisms anddifferent types of libraries (i.e. different fusion proteins, differentdelivery systems) will yield a wide numerical range of protective genes.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to the methodsand compositions and in the steps or in the sequence of steps of themethods described herein without departing from the concept, spirit andscope of the invention. More particularly, it will be apparent that themethods and concepts have a very wide range of application to virtuallyall categories of human and non-human pathogens and while demonstratedwith two pathogens could be applied to any foreign DNA, includingcancer. All such similar applications and substitutes apparent to thoseskilled in the art in view of the present disclosure and knowntechniques are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

The following references as well as those cited in the specification areincorporated in pertinent part by reference herein for reasons cited inthe text.

REFERENCES

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 8                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #                 22AAG TA                                                    - (2) INFORMATION FOR SEQ ID NO:2:                                            - 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    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #          32      ATCC TAAGTAAGTA AG                                         - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Thr Gly Gln Ile Leu Asp Pro Lys                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #           30     CCTA AGTAAGTAAG                                            - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Thr Gly Gln Ile Leu Asp                                                     1               5                                                             __________________________________________________________________________

What is claimed is:
 1. A composition comprising one or more antigensexpressed from DNA segments obtained from a sib library of a clonedexpression library prepared from a pathogen DNA or RNA wherein said oneor more antigens produce a protective response to said pathogen in avertebrate animal.
 2. The composition of claim 1 which is comprised in apharmaceutically acceptable vehicle.
 3. The composition of claim 1wherein the pathogen DNA is Mycobacterium tuberculosis, Mycoplasmapulmonis, Chlamydia or Listeria monocytogenes.
 4. The composition ofclaim 1 wherein the vertebrate animal is a human.
 5. The composition ofclaim 1 wherein the pathogen is HIV.
 6. The composition of claim 1wherein the pathogen is bacterial, viral, fungal or parasitic.